Recombinant DNA technology has permitted the expression of heterologous protein in host cells such as E. coli bacteria. In the case of somatotropin, a growth hormone, the protein is sequestered in refractile bodies within the cytoplasm of the host cells. The refractile bodies may be recovered from the host cell culture by disrupting the cell so as to release the refractile bodies, and thereupon collecting the refractile bodies as a solid pellet by differential centrifugation. The refractile bodies are solubilized in an aqueous solution of a suitable chaotropic agent such as urea or guanidine hydrochloride at an alkaline pH, generally in the range of 10-12. The solubilized proteins are subsequently naturized by contact with a mild oxidizing agent to form intramolecular disulfide bonds and refold or return the protein to its biologically active native conformation. Methods for the solubilization and the naturation of somatotropin protein produced by E. coli bacteria using recombinant DNA technology are described in U.S. Pat. No. 4,511,502 and U.S. Pat. No. 4,652,630, each of which is incorporated herein by reference.
The refold solution obtained from the naturation step consists of an aqueous solution of somatotropin monomers, dimers and higher oligomers, with residues and other debris from the host cells. Of these, the somatotropin monomer is the desired biologically active agent and must be recovered in a highly purified form suitable for administration by injection to the target animal.
Purification of proteins is a common problem in biotechnology, and several methods to accomplish such purification have been developed. A literature survey entitled "Protein Purification: The Right Step at the Right Time" by J. Bonnerjea et al, reported in Biotechnology, Volume 4, pages 955-958 (November 1986) identified ten common methods for protein purification with ion exchange chromatography, affinity chromatography and gel filtration being the three most popular. As reported in this article, an average of four purification steps were necessary to purify proteins to homogeneity with an overall yield of 28% and a purification factor of 6,380. More than half the purification schemes were reported to include precipitation, generally as the first or second step after homogenation. The most popular scheme of purification was homogenization followed by precipitation, ion exchange chromatography, affinity separation and finally gel filtration. Precipitation was reported to produce an average purification factor of threefold.
Biologically active products derived from microbial sources and intended for parenteral use in animals must be substantially free from contaminating substances such as pyrogens, native microbial proteins and nucleic acids if they are not to initiate adverse reactions such as allergic response, fever and other side effects when administered. As reported in "Downstream Processing of Fermentation Products by Chromatography" by John M. Curling et al, American Biotechnology Laboratory, pages 33-37 (1983), traditional purification techniques such as precipitation technology which has been used since the 1950's for plasma fractionation are unable to achieve the high levels of purity that are required for recombinant DNA produced products. Of the high resolution purification techniques available to the biotechnology industry, chromatography is considered to be the method of choice at both research and production levels. The term "chromatography" includes the specific methods of ion-exchange, gel filtration, affinity chromatography, hydrophobic chromatography, and chromatofocusing. Chromatofocusing is a technique which separates proteins according to their isoelectric points in a resin bed having a pH gradient created by titrating a chromatographic gel with a specially prepared buffer.
Chromatography is reported to be the method of choice for commercial recovery of ultrapure human proteins and hormones from bacterial homogenates for therapeutic applications. The chromatographic purification is preferably preceded by an initial fractionation step such as protein precipitation or liquid-liquid partitioning to enrich the chromatographic feed stream and improve efficiencies as discussed in "Trends in Downstream Processing of Proteins and Enzymes" by P. Dunnill, Process Biochemistry, pages 9-13 (October 1983).
An example of the use of chromatography in the purification of prochymosin expressed from E. coli through DNA recombinant technology is described in "Synthesis of Calf Prochymosin (prorennin) in Escherichia coli" by J. M. Emtage et al, Biochemistry, Volume 80, pages 3671-3675 (June 1983). This reference also mentions that the majority of E. coli proteins (about 90%) precipitated under acidic conditions (pH 6.3) and could be removed by centrifugation.
Separation of proteins by precipitation techniques is discussed in "The Molecular Basis of Cell Structure and Function" by Albert L. Lehninger, Biochemistry, 2nd Edition, The John Hopkins School of Medicine (1975). This text explains that proteins in solution show profound changes in solubility as a function of pH, ionic strength, temperature and dielectric properties of the solvent, and that a protein is least soluble at its isoelectric pH, i.e., the pH at which the molecule has no net electric charge. Under such conditions, the protein molecules tend to coalesce and precipitate. Some proteins are virtually insoluble at their isoelectric pH and, as reported in this reference, where different proteins in solution have different isoelectric pH values, they can often be separated from each other by the method of isoelectric precipitation.
Purification by fractional precipitation based on pH adjustment is further discussed in Biochemical Engineering Fundamentals, Bailey and Ollis, 2nd Edition, pages 745-749 (1986). The method is suggested for separating proteins with different isoelectric points (pI), since at a given pH, proteins with the nearest pI will tend to precipitate, other things such as molecular weight being equal. By varying pH, fractions containing different proteins may be separated.
The solubility of proteins is affected by salt concentration and temperature of the solvent, and the resolution of precipitation fractionation may sometimes be improved by using salting out techniques and/or low temperatures to further reduce or enhance protein solubility. Isoelectric precipitation of low solubility proteins may also be improved by inclusion of a solute such as ethanol or acetone or an organic polymer such as polyethylene glycol in an aqueous medium. These techniques are discussed in Protein Purification: Principles and Practice by R. K. Scopes, 2nd Edition, pages 41-71 (1987). A classic example of the use of a salting out technique in a precipitation fractionation process mentioned in this reference is the one-step purification of glyceraldehyde phosphate dehydrogenase from rabbit muscle by precipitation at a pH above 7.5 and in an aqueous solution containing 3.2M ammonium sulfate.
As seen from the above discussion, precipitation fractionation is an old and well-known technique for obtaining some degree of separation and refinement of proteins in solution, and is an accepted method for making gross separations in order to enrich a feedstock for further processing and final purification by high resolution techniques such as chromatography. While specific conditions under which the precipitation fractionation is conducted can affect yield and purity of the resulting protein, the precipitation technique has not heretofore been utilized for final purification of proteins intended for parenteral administration by injection into target animals. Bovine somatotropin produced by recombinant DNA methodology for use in beef and dairy cattle, and the corresponding porcine somatotropin for use in raising hogs, have generally been purified by conventional chromatographic techniques. One such technique which involves subjecting impure protein stock solution to reverse phase purification on a macroporous acrylate ester copolymer resin support followed by elution with an organic diluent in aqueous solution at a pH between about 7 and 9 is described in U.S. Pat. No. 4,612,326.
The following additional U.S. patents are of interest for their disclosure of various protein purification techniques.
U.S. Pat. No. 3,239,418 describes a two-stage precipitation process followed by ion exchange to purify a pituitary growth hormone promoter.
U.S. Pat. No. 4,054,557 describes the purification of growth promoting polypeptides by gel filtration.
U.S. Pat. No. 4,426,323 describes a two-stage desalting and pH precipitation process wherein impurities are first removed by precipitation, following which the desired protein is precipitated and recovered.
U.S. Pat. No. 4,451,487 describes a process for the purification of biologically active proteins by the addition of a selected precipitation agent which promotes the precipitation of the desired protein while leaving impurities in solution.
U.S. Pat. No. 4,569,794 describes the purification of biologically active polypeptides and proteins by chromatographic techniques.
U.S. Pat. No. 4,617,376 describes a process for the purification of glucagon from a glucagon salt cake by hydrophobic chromatography.
Prior art chromatographic processes for the purification of proteins, including somatotropin proteins, are complex and expensive. Precipitation techniques, while relatively simple and inexpensive to operate, have not produced the yields and resolution required for products intended for parenteral use.
It is accordingly an object of the present invention to provide a method for purifying somatotropin proteins produced by recombinant DNA methodology.
It is a further object of this invention to provide a process for separating somatotropin monomers from somatotropin dimers and higher oligomers contained in solution.
It is a yet further object of this invention to provide a method for the isolation and recovery of somatotropin monomers from solutions containing somatotropin monomers and oligomers with residual host cell proteins and other contaminants.
A still further object of this invention is to provide an economical precipitation fractionation process for the recovery of high purity somatotropin monomers from the refold solution of the naturation step in a recombinant DNA process for the manufacture of somatotropin.
These and other objects and advantages of the present invention will be evident to those skilled in the art from the following description and examples.